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. 2016 Apr;349(4):233-41.
doi: 10.1002/ardp.201500440. Epub 2016 Mar 7.

Synthesis of Arylazide- and Diazirine-Containing CrAsH-EDT2 Photoaffinity Probes

Shameem S Syeda  1   2 , Daren Rice  2   3 , Derek J Hook  1   2 , Leslie L Heckert  2   3 , Gunda I Georg  1   2
Affiliations

Synthesis of Arylazide- and Diazirine-Containing CrAsH-EDT2 Photoaffinity Probes

Shameem S Syeda et al. Arch Pharm (Weinheim). 2016 Apr.

Erratum in

Abstract

Two photo-crosslinking biarsenical (CrAsH-EDT2 )-modified probes were synthesized that are expected to be useful tools for tetracysteine-labeled proteins to facilitate the co-affinity purification of their DNA binding sequences and interacting proteins. In addition, improvements for the synthesis of CrAsH-EDT2 and N(1) -(4-azido-2-nitrophenyl)hexane-1,6-diamine are reported. Both photoprobes effectively entered HeLa cells (and the nucleus) and were dependent on the tetracysteine motif in recombinant DMRT1 (doublesex and Mab3-related transcription factor) to induce fluorescence, suggesting that their crosslinking abilities can be exploited for the identification of nucleic acids and proteins associated with a protein of interest.

Keywords: Biarsenical probe; Fluorescence; Photoaffinity probes; Tetracysteine-tagged recombinant DMRT1.

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Figures

Figure 1
Figure 1
Structures of non‐fluorescent FlAsH‐EDT2 and CrAsH‐EDT2 and green fluorescent FlAsH‐EDT2 and CrAsH‐EDT2 complexed with Cys‐Cys‐Xaa‐Xaa‐Cys‐Cys and structures of TRAP, azide‐TRAP (1) and diazirine‐TRAP (2) photo‐crosslinking biarsenical probes.
Scheme 1
Scheme 1
Preparation of affinity probes azide‐TRAP (1) and diazirine‐TRAP (2).
Scheme 2
Scheme 2
Preparation of phenylazide 9.
Scheme 3
Scheme 3
Synthesis of diazirin 13.
Figure 2
Figure 2
Live cell imaging with FlAsH‐EDT2, azide‐TRAP (1) and diazirine‐TRAP (2). HeLa cells were transfected with an expression vector for DMRT1 containing the optimized biarsenical binding tetracysteine motif on its amino terminus (FLN‐DMRT1). Cells were then treated with either FlAsH‐EDT2 (A and D), compound 1 (B and E) or compound 2 (C and F). The images A–F were generated from the FLN‐DMRT1 transfected cells with the background subtracted. Images are 200× (A–C) or enlargements of the boxed areas (D–F).
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